Gene editing method for regulating and controlling nitrogen utilization rate of rice and regenerated rice plant

Abstract

The invention discloses a gene editing method for regulating and controlling the nitrogen utilization rate of rice and a regenerated rice plant, and relates to the technical field of agricultural biology. The gene editing method for regulating and controlling the nitrogen utilization rate of the rice comprises the following steps: selecting a key gene of nitrogen metabolism of the rice as a target gene; determining a target spot in a promoter region of the target gene; constructing a repair donor template containing a nitrate response element based on the sequences on the two sides of the target spot; wherein the nitrate response element is derived from a promoter region of rice nitrite reductase, and can respond to a nitrate signal to enhance the expression of a downstream gene; jointly delivering the repaired donor template and a CRISPR / Cas9 carrier to the rice callus through gene editing, and culturing; after the culture is completed, screening to obtain a regenerated rice plant with the nitrate response element accurately inserted in the genome. According to the application, the nitrate response element is inserted into the promoter region of the nitrogen metabolism key gene to activate gene expression induced by nitrate, so that nitrogen utilization of rice is enhanced, and regenerated rice plants with high nitrogen utilization efficiency are obtained.

Description

Technical Field

[0001] This application relates to the field of agricultural biotechnology, and particularly to a gene editing method for regulating nitrogen use efficiency in rice and a regenerated rice plant. Background Art

[0002] Nitrogen fertilizers are the most widely used type of fertilizer in agricultural production. Nitrogen fertilizers and nitrogen-containing compound fertilizers account for more than 60% of the total fertilizer application. The application of nitrogen fertilizers can significantly increase the yield and quality of rice. However, the current nitrogen fertilizer supply in China is seriously excessive. As the most important food crop in China, only 30%-50% of the applied nitrogen fertilizer can be utilized in rice, resulting in a large amount of nitrogen leaching, serious soil acidification, and water eutrophication. Therefore, improving the nitrogen use efficiency of rice is an important way to achieve sustainable agricultural production. At present, the breeding of rice varieties with high nitrogen use efficiency has received extensive attention globally, mainly through selective breeding and transgenic breeding methods. In the selective breeding method, although some rice varieties have shown relatively high nitrogen use efficiency, the genetic sources of these excellent traits are still limited, restricting the diversity of breeding materials. The lack of diversity leads to limitations in the selection space during the breeding process. In addition, the long breeding cycle is also a major problem. Traditional rice breeding usually requires long-term screening and repeated field trials to ensure the stability and adaptability of the varieties. During the breeding process of high nitrogen use efficiency, the breeding cycle is even longer. The transgenic breeding method is difficult to achieve fine regulation of the expression level, and the obtained plants are cumbersome to approve due to the presence of foreign DNA. Based on this, this application proposes a gene editing method for regulating nitrogen use efficiency in rice and a regenerated rice plant. Based on this, this application proposes a gene editing method for regulating nitrogen use efficiency in rice and a regenerated rice plant. Summary of the Invention

[0003] The main object of this application is to provide a gene editing method for regulating nitrogen use efficiency in rice and a regenerated rice plant, aiming to solve the technical problem of low nitrogen use efficiency in existing rice varieties.

[0004] To achieve the above object, this application proposes a gene editing method for regulating nitrogen use efficiency in rice, as Figure 1 shown, including the following steps:

[0005] Select a key gene for nitrogen metabolism in rice as the target gene;

[0006] Determine the target site in the promoter region of the target gene;

[0007] Based on the sequences on both sides of the target site, construct a repair donor template containing a nitrate response element; wherein the nitrate response element is derived from the promoter region of rice nitrite reductase and can respond to nitrate signals to enhance the expression of downstream genes;

[0008] After co-delivering the repair donor template and the CRISPR / Cas9 vector to rice calli through gene editing, culture is carried out.

[0009] After the culture is completed, regenerated rice plants with the nitrate response element inserted into their genomes are screened and obtained.

[0010] Optionally, in the step of selecting the key genes of rice nitrogen metabolism as target genes, the target genes are at least one of the GRF4 target gene and the GS1;2 target gene.

[0011] Optionally, the gene sequence of the promoter region of the GRF4 target gene is as shown in Seq ID NO.3; the gene sequence of the promoter region of the GS1;2 target gene is as shown in Seq ID NO.4.

[0012] Optionally, the step of determining the target site in the promoter region of the target gene includes:

[0013] Using the dual-luciferase reporter gene assay, the promoter fragments of the target gene are cloned in segments, multiple reporter gene vectors with the nitrate response element inserted at different positions are constructed, the expression enhancement effect is verified through protoplast transformation and nitrate induction experiments, and the position capable of enhancing the expression of downstream genes is determined as the target site;

[0014] Among them, the target site is within the range of 0.1 kb - 1.0 kb near the transcription start site of the target gene.

[0015] Optionally, the nitrate response element is 4 tandem copies of the NRE cis-acting element, and the gene sequence of the nitrate response element is as shown in Seq ID NO.1.

[0016] Optionally, in the step of constructing a repair donor template containing a nitrate response element based on the sequences on both sides of the target site, the homologous arms of the repair donor template match the sequences on both sides of the target site, the repair donor template uses a linear DNA fragment with chemical modifications, and the repair donor template is prepared by amplifying with a pair of primers modified with 5' phosphate and terminal 3-base thiolation.

[0017] Optionally, in the step of co-delivering the repair donor template and the CRISPR / Cas9 vector to rice calli through gene editing, the CRISPR / Cas9 vector is obtained by connecting the Ubiquitin promoter - Cas9 protein - Nos terminator, the U3 promoter - sgRNA, and the hygromycin resistance screening marker gene in series through the GoldenGate method to the pCam1300 vector backbone.

[0018] Optionally, the gene sequence of the CRISPR / Cas9 vector is as shown in Seq ID NO.2.

[0019] Optionally, in the step of co-delivering the repair donor template and the CRISPR / Cas9 vector to rice callus by gene editing, the gene gun transformation method is used to co-deliver the repair donor template and the CRISPR / Cas9 vector to rice callus;

[0020] The parameters of the gene gun transformation method include: the gold powder particle size is 0.5μm - 0.7μm; the helium pressure is 1100psi - 1350psi; the target distance is 5cm - 7cm; the CRISPR / Cas9 vector and the repair donor template are mixed in a molar ratio of 1:(5 - 10).

[0021] The present application also provides a regenerated rice plant obtained by the above gene editing method for regulating rice nitrogen use efficiency. In the genome of the regenerated rice plant, the promoter region of the GRF4 gene and / or the promoter region of the GS1;2 gene contains an inserted nitrate response element.

[0022] The present application at least includes the following beneficial effects:

[0023] In the present application, four tandem cis-acting element (4×NRE) sequences are inserted into the promoter regions of two important nitrogen metabolism-related genes (GRF4, GS1;2) to activate gene expression under nitrate induction, thereby enhancing the nitrogen utilization of rice, that is, enhancing the expression of target genes under the condition of applying nitrogen fertilizer in the field, obtaining regenerated rice plants with high nitrogen use efficiency, thereby reducing the amount of nitrogen fertilizer applied and reducing the risks of nitrogen leaching, soil acidification and water eutrophication, meeting the requirements of sustainable development of green agriculture;

[0024] The present application uses gene gun to deliver CRISPR / Cas9 and the repair donor template, and the insertion efficiency is as high as 17.54% (GS1;2 target site), and the present application uses a chemically modified donor template (5'-phosphate and terminal 3-base thiophosphate modification), which improves the stability and insertion accuracy of the repair donor template;

[0025] The 4×NRE inserted in the present application can significantly enhance the expression of downstream genes, especially in the presence of nitrate, and when the insertion position of 4×NRE is within the range of 100bp - 400bp near the transcription start site, the gene activation effect is the best, and the expression level can be increased by up to 60 times (GS1;2) to 6000 times (GRF4); the transcription levels of GRF4 and GS1;2 in the obtained regenerated rice plants are significantly increased under both low-nitrogen and high-nitrogen conditions, thereby enhancing their nitrogen response ability;

[0026] This application provides a new strategy for high nitrogen use efficiency breeding in rice, which can be directly applied to cultivated varieties (such as Ningjing 7), shortening the breeding cycle, and significantly improving the agronomic traits such as the yield and phenotype of the ratoon rice plants. At the same time, the method of this application also provides a technical reference for the precision breeding of other crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the structures shown in these drawings.

[0028] Figure 1 It is a flowchart of the gene editing method for regulating the nitrogen use efficiency of rice according to the embodiment of the present application;

[0029] Figure 2 It is a schematic diagram of the insertion sites of the NRE fragment in the GRF4 promoter region and the GS1;2 promoter region according to the embodiment of the present application;

[0030] Figure 3 It is a comparison result diagram of the activities of different insertion sites of the NRE fragment in the GRF4 promoter region and the GS1;2 promoter region according to the embodiment of the present application; among them, (a) is the comparison result diagram of the activities of different insertion sites of the NRE fragment in the GRF4 promoter region, and (b) is the comparison result diagram of the activities of different insertion sites of the NRE fragment in the GS1;2 promoter region;

[0031] Figure 4 It is a schematic diagram of the site-directed insertion of the NRE into the GRF4 promoter region and the GS1;2 promoter region according to the embodiment of the present application;

[0032] Figure 5 It is a sequencing result diagram of the family plants GRF4-NRE#7, GRF4-NRE#8, GS1;2-NRE#1 and GS1;2-NRE#5 according to the embodiment of the present application;

[0033] Figure 6 It is a relative expression quantity result diagram of genes in the leaf tissues of the GRF4-NRE family plants according to the embodiment of the present application under low nitrogen and high nitrogen culture conditions; among them, (c) is the relative expression quantity result diagram of genes in the leaf tissues of the GRF4-NRE family plants under low nitrogen culture conditions, and (d) is the relative expression quantity result diagram of genes in the leaf tissues of the GRF4-NRE family plants under high nitrogen culture conditions;

[0034] Figure 7Results graph of the relative expression levels of genes in the root tissues of GRF4-NRE family plants according to the embodiments of the present application under low-nitrogen and high-nitrogen culture conditions; among them, figure (e) is the results graph of the relative expression levels of genes in the root tissues of GRF4-NRE family plants under low-nitrogen culture conditions, and figure (f) is the results graph of the relative expression levels of genes in the root tissues of GRF4-NRE family plants under high-nitrogen culture conditions;

[0035] Figure 8 Results graph of the relative expression levels of genes in the leaf tissues of GS1;2-NRE family plants according to the embodiments of the present application under high-nitrogen and low-nitrogen culture conditions; among them, figure (g) is the results graph of the relative expression levels of genes in the leaf tissues of GS1;2-NRE family plants under low-nitrogen culture conditions, and figure (h) is the results graph of the relative expression levels of genes in the leaf tissues of GS1;2-NRE family plants under high-nitrogen culture conditions;

[0036] Figure 9 Results graph of the relative expression levels of genes in the root tissues of GS1;2-NRE family plants according to the embodiments of the present application under high-nitrogen and low-nitrogen culture conditions; among them, figure (i) is the results graph of the relative expression levels of genes in the root tissues of GS1;2-NRE family plants under low-nitrogen culture conditions, and figure (j) is the results graph of the relative expression levels of genes in the root tissues of GS1;2-NRE family plants under high-nitrogen culture conditions;

[0037] Figure 10 Phenotype schematic diagram of GRF4-NRE#8 family plants according to the embodiments of the present application;

[0038] Figure 11 Results graph of the effective tiller number of GRF4-NRE family plants according to the embodiments of the present application;

[0039] Figure 12 Results graph of the number of grains per panicle of GRF4-NRE family plants according to the embodiments of the present application;

[0040] Figure 13 Results graph of the 1000-grain weight of GRF4-NRE family plants according to the embodiments of the present application;

[0041] Figure 14 Phenotype schematic diagram of GS1;2-NRE#24 family plants according to the embodiments of the present application;

[0042] Figure 15 Results graph of the effective tiller number of GS1;2-NRE family plants according to the embodiments of the present application;

[0043] Figure 16 Results graph of the number of grains per panicle of GS1;2-NRE family plants according to the embodiments of the present application;

[0044] Figure 17 The thousand-grain weight result chart of the GS1; 2-NRE family plants described in the embodiments of the present application.

[0045] The realization, functional features and advantages of the present application will be further described in conjunction with the embodiments and with reference to the accompanying drawings. Detailed implementation manners

[0046] Next, the technical solutions in the embodiments of the present application will be clearly and completely described with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts shall fall within the protection scope of the present application.

[0047] Sequence listing description (the sequence listing content is provided separately):

[0048] The gene sequence of the nitrate response element in the embodiments of the present application is shown in Seq ID NO.1;

[0049] The gene sequence of the CRISPR / Cas9 vector in the embodiments of the present application is shown in Seq ID NO.2;

[0050] The gene sequence of the promoter region of the target gene GRF4 in the embodiments of the present application is shown in Seq ID NO.3;

[0051] The gene sequence of the promoter region of the target gene GS1; 2 in the embodiments of the present application is shown in Seq ID NO.4.

[0052] Example 1 Target gene selection and insertion target point design

[0053] 1.1 Target gene selection

[0054] Select the key genes GRF4 and / or GS1; 2 of rice nitrogen metabolism as the target genes.

[0055] Among them, the gene sequence of the promoter region of the GRF4 target gene is shown in Seq ID NO.3;

[0056] The gene sequence of the promoter region of the GS1; 2 target gene is shown in Seq ID NO.4.

[0057] 1.2 Insertion target point design

[0058] In this application, the target design mainly uses the CRISPR-P 2.0 online website (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR) developed by the team at Huazhong Agricultural University. The promoter sequence of the target gene is input into the website for target site analysis, and specific sequences with moderate GC content and low potential off-target rate are selected as target sites from the obtained target site analysis results.

[0059] Then, the Cas-OFFinder online website (http: / / www.rgenome.net / cas-offinder / ) is used to predict potential off-target sites. The 20bp target sequence is input, and the conditions of ≤2bp mismatch with the Oryza sativa (Osv4)-Rice reference genome and ≤1bp DNA or RNA bulge are selected for retrieval. After obtaining the specific chromosomal positions of the potential off-target sites, the sequence information of the off-target sites is obtained using GenBank in the NCBI (National Center for Biotechnology Information) database.

[0060] 1.3 Determine the optimal position of the inserted target through the dual-luciferase reporter gene experiment.

[0061] 1.3.1 Experimental materials

[0062] Rice varieties: Japonica rice Nipponbare; Ningjing 7 (N7).

[0063] Vector system:

[0064] Reporter vector: pGreenII-LUC (containing FLUC and RLUC reporter genes), which has been stored in the laboratory for a long time. The RLUC gene expression is initiated by the CaMV35S promoter, and there is a multiple cloning site in front of the coding region of the FLUC gene;

[0065] Donor vector: pUC57-4×NRE (containing the 4×NRE tandem sequence, i.e., the sequence shown in Seq ID NO.1), synthesized by GenScript Corporation (Nanjing).

[0066] Reagents:

[0067] Restriction endonucleases BamHI, HindIII, XhoI;

[0068] High-fidelity DNA polymerase Phanta Max Super-Fidelity DNA Polymerase;

[0069] T4 DNA ligase;

[0070] PEG4000 transformation solution (40% w / v, containing 0.2 M mannitol).

[0071] 1.3.2 Experimental procedures

[0072] 1.3.2.1 Construction of dual luciferase vectors

[0073] Using pUC57-4×NRE as a template, the NRE fragment was amplified by PCR with M13F / M13R primers;

[0074] PCR conditions: 98°C for 30 s; 35 cycles (98°C for 10 s, 60°C for 15 s, 72°C for 20 s); 72°C for 5 min;

[0075] After detection by 1% agarose gel electrophoresis and gel extraction and recovery, the NRE fragment with sticky ends was obtained by treatment with restriction enzymes BamHI and HindIII;

[0076] pGreenII-LUC was also treated with restriction enzymes BamHI and HindIII to obtain a linearized vector with the same sticky ends as the NRE fragment, which was detected by 1% agarose gel electrophoresis and gel extraction and recovery;

[0077] The NRE fragment and the linearized vector were mixed at a molar concentration of 1:1, ligated overnight at 25°C using T4 ligase, and then transformed into Escherichia coli. The positive clones were identified and verified by sequencing to obtain the pGreenII-NRE-LUC vector. The insertion site of the NRE fragment is as Figure 2 shown;

[0078] The promoter fragments (~2000 bp) of GRF4 and GS1;2 genes were amplified from the genome of Ningjing 7, and the full-length promoters of GRF4 and GS1;2 genes were treated with restriction enzymes XhoI and HindIII;

[0079] The promoter fragments with sticky ends were recovered and mixed with the linearized pGreenII-LUC vector with the same sticky ends at a molar concentration of 1:1, ligated overnight at 25°C using T4 ligase, and then transformed into Escherichia coli. The positive clones were identified and verified by sequencing to obtain the pGreenII-GRF4-LUC vector and the pGreenII-GS1;2-LUC vector respectively.

[0080] 1.3.2.2 Transformation of rice protoplasts

[0081] Preparation of rice protoplasts: Take 14-day-old Nip seedlings aseptically cultured in MS medium, select the shoot meristem tissue near the root, chop it up and let it stand in the dark in 0.6M mannitol solution for 10 min; discard the mannitol solution, transfer the chopped seedlings to a conical flask containing enzyme solution, evacuate with a vacuum pump in the dark for 30 min; place it in a shaker at 28 °C and 70 rpm, and enzymatically digest in the dark for 6 h; then add W5 solution with the same volume as the enzyme solution to terminate the reaction, and gently shake at 28 °C and 70 rpm for 5 min; filter the liquid in the conical flask through a 45-μm cell sieve into a 50-mL round-bottom centrifuge tube, and then wash the chopped seedlings with a certain volume of W5 solution and filter again; centrifuge at 28 °C and 250×g for 3 min, and discard the supernatant; resuspend the precipitate with 10 mL - 15 mL W5 solution, centrifuge at 250×g for 3 min, and discard the supernatant; add a certain volume of MMG solution to resuspend the protoplast cells.

[0082] PEG transformation: Sequentially add 20 μL of 1 μg / μL dual-luciferase vector, 200 μL of protoplast cells, and 220 μL of PEG solution to a sterile 2-mL centrifuge tube, gently mix and let it stand in the dark for 20 min - 30 min; after the transformation is completed, add 880 μL of W5 solution along the tube wall, slowly invert and mix; centrifuge at 250×g for 2 min, and discard the supernatant; add 1 mL of WI solution to the centrifuge tube and culture in the dark at 28 °C for 20 h.

[0083] 1.3.2.3 Induction and detection

[0084] 18 h after transforming rice protoplasts, add 0.2 mM KNO3 (experimental group) or KCl (control group) to the original WI solution, and measure the expression level of dual-luciferase 2 h later. Use the Promega dual-luciferase kit to detect the activities of Renilla luciferase (Rluc) and firefly luciferase (Fluc) in the samples respectively, and calculate the relative activity: the Fluc / Rluc ratio.

[0085] 1.3.3 Experimental results

[0086] The comparison results of the activities of different insertion sites in the GRF4 promoter region and the GS1;2 promoter region are as Figure 3 shown.

[0087] As Figure 3 can be seen, the ratio of the expression level of dual-luciferase transcribed by the original gene promoter without the cis-acting element NRE is not induced by KNO3 or KCl treatment. However, after inserting NRE at a certain position in any promoter, the ratio of the expression level of dual-luciferase will be affected by different treatments, and the ratio will increase under nitrate treatment, indicating that after inserting the nitrate response element in the GRF4 promoter region and the GS1;2 promoter region in this application, it can respond to nitrate conditions and further activate the expression of downstream genes.

[0088] Moreover, when the NRE is inserted at a relatively far position from the transcription start site (TSS), the ability of the NRE to enhance downstream gene expression is weak, but there is still a certain activation effect; inserting the NRE at the position closest to the TSS can obtain the best significant activation effect. Preferably, the optimal insertion site is the region of 100 bp - 400 bp near the TSS of the target gene.

[0089] Example 2: Gene gun-mediated genetic transformation of rice

[0090] 2.1 Construction of CRISPR / Cas9 vector

[0091] 2.1.1 sgRNA design

[0092] Design sgRNA target sites for the GRF4 promoter region (the sequence shown in Seq ID NO.3): 5'-CTGCCGAAGTGGAACTACGANGG-3';

[0093] Design sgRNA target sites for the GS1;2 promoter region (the sequence shown in Seq ID NO.4): 5'-TGTCTCTGAATGATACTTGCC-3';

[0094] Verify the target site specificity through the CRISPR-P 2.0 online tool to ensure that the off-target rate < 0.1%.

[0095] 2.1.2 GoldenGate assembly

[0096] Connect the Ubiquitin promoter - Cas9 protein - Nos terminator, U3 promoter - sgRNA, and hygromycin resistance screening marker gene in series through the GoldenGate method to the pCam1300 vector backbone to obtain the CRISPR / Cas9 vector, and its gene sequence is as shown in Seq ID NO.2;

[0097] The Goldengate reaction program is: 50 cycles (37°C for 3 min, 16°C for 2 min); 37°C for 30 min; 65°C for 20 min; 12°C for 10 min;

[0098] The Goldengate reaction system is shown in Table 1 below.

[0099] Table 1

[0100] Reagent Volume BsaI-HF Restriction Endonuclease 0.5 μL T4 DNA Ligase 0.5 μL 10×rCutsmart Buffer 2 μL 10×T4 DNA Ligase Buffer 2 μL Vector DNA 100 ng Insert DNA 50 ng - 100 ng <![CDATA[ddH2O]]> Add to 20 μL

[0101] 2.2 Preparation of repair donor template

[0102] Design a linear donor template containing 4×NRE (as shown in Seq ID NO.1). The linear form can increase the transformation concentration. Add 50bp homologous arms at both ends (matching the flanking sequences of the target. In this example, the target position is as Figure 4 shown). Prepare it by amplifying with a pair of primers modified with 5'-phosphate and 3'-terminal phosphorothioate modification. It has phosphorothioate modification at both ends, which can prevent cell degradation to a certain extent and improve the efficiency of fragment insertion. Use a high-fidelity DNA polymerase for amplification, then detect by 1.5% agarose gel electrophoresis, and cut and recover the gel.

[0103] 2.3 Rice callus culture

[0104] Remove the glumes from the seeds of Ningjing 7, and select plump high-quality seeds without obvious disease spots or plaques. Wash them with 75% ethanol by vortex for 1 min, then soak and disinfect the seeds with 30% sodium hypochlorite on a shaker for 30 min, rinse them with sterile pure water 3 - 4 times, and finally dry the surface moisture of the seeds with sterile absorbent paper; Place 10 seeds evenly on each plate of the induction medium (MS medium + 2 mg / L 2,4-dichlorophenoxyacetic acid), and culture them in the dark at 26°C for 25 days to induce rice callus; Select compact light yellow embryogenic callus onto the subculture medium (with the same composition as the induction medium) and culture it in the dark at 26°C for 7 days; Transfer the subcultured callus to the high-osmotic medium (MS medium + 0.4 M mannitol) more than 4 h before gene gun bombardment to increase the osmotic pressure.

[0105] 2.4 Gene gun bombardment

[0106] Gold powder treatment: After washing 0.6μm gold powder with absolute ethanol, resuspend it in 50% glycerol;

[0107] DNA embedding: Mix the CRISPR / Cas9 vector and the repair donor template at a molar ratio of 1:1, and add 0.1 M spermidine and 2.5 M CaCl2 for precipitation;

[0108] Bombardment conditions: Helium pressure 1100 psi, target distance 6 cm, vacuum 28 inches Hg.

[0109] Gene gun model: PDS-1000 / He bench-top gene gun from Bio-Rad company.

[0110] 2.5 Genetic transformation and plant regeneration

[0111] After bombardment, it was still cultured overnight in hypertonic medium, and then transferred to induction medium again the next day for 3 days of recovery without changing the orientation of the callus during transfer; after three days, it was transferred to a selection medium containing 50 mg / L hygromycin and cultured at 26 °C for 25 days; the dense light yellow small embryogenic callus newly grown on the selection medium was transferred to differentiation medium and cultured at 26 °C under a photoperiod of 16 h light / 8 h dark for about 20 days. When the newly grown shoots were about 3 cm long, the differentiated callus was transferred to rooting medium for rooting and cultured for about 15 days under the same temperature and photoperiod conditions; the medium on the roots was washed off, and the regenerated plants were acclimatized under high temperature and high humidity conditions to obtain T0 plants with 4×NRE fragment insertion. The schematic diagram of the site-directed insertion of 4×NRE in the genome of T0 plants is shown in Figure 4 as shown.

[0112] Example 3 Molecular Identification and Expression Analysis

[0113] 3.1 PCR Identification

[0114] Due to the loss of some plants during the transplantation of T0 plants, 3 GRF4-NRE families and 7 GS1;2-NRE families were finally obtained. In this example, plants from GRF4-NRE#7, GRF4-NRE#8, GS1;2-NRE#1 and GS1;2-NRE#5 families were selected for sequencing.

[0115] Plants from GRF4-NRE#7, GRF4-NRE#8, GS1;2-NRE#1 and GS1;2-NRE#5 families were amplified and identified using flanking primers of the target site respectively. The sequencing results are shown in Figure 5 as shown. The insertion efficiency of GRF4 and GS1;2 target sites is shown in Table 2 below.

[0116] Table 2

[0117]

[0118] As can be seen from Table 2, the insertion efficiency of the NRE fragment-containing is 4.49% at the GRF4 target site and 17.54% at the GS1;2 target site; as Figure 5 can be seen, compared with the wild-type band, the amplification product containing the inserted 4×NRE fragment shows a larger fragment length, and the sequencing shows seamless insertion.

[0119] 3.2 Fluorescent Quantitative PCR Analysis of Gene Expression

[0120] Sample treatment: T1 generation plants were hydroponically cultured for 14 days and treated with low nitrogen (0.144 mM) and high nitrogen (1.44 mM) for 24 h respectively.

[0121] RNA extraction and reverse transcription: Total RNA was extracted from leaf and root tissues using the TRIzol method and reverse transcribed into cDNA using HiFiScript.

[0122] qPCR conditions: SYBR Green premix, ABI QuantStudio 5 instrument, program: 95°C for 3 min; 40 cycles (95°C for 15 s; 60°C for 30 s).

[0123] Reference gene: OsActin1 (primers: 5'-GCCAACAGAGAGAAGATGAC-3' / 5'-ACCAGCAAGGTCAAGACGAA-3').

[0124] Result analysis:

[0125] Using NingJing7 as the control group without inserted fragments, the transcriptional levels of genes in the leaf and root tissues of the T1 generation plants of the GRF4-NRE and GS1;2-NRE families were tested under high-nitrogen and low-nitrogen culture conditions. The transcriptional levels were represented by the relative expression levels of the reference gene Actin1. The relative expression level of NingJing7 was 1, and the relative expression levels of the T1 generation plants of the GRF4-NRE and GS1;2-NRE families were as Figures 6 - 9 shown.

[0126] As Figures 6 - 7 can be seen, under high-nitrogen and low-nitrogen conditions, 4×NRE was inserted into the upstream regulatory sequence of GS1;2, increasing its expression level in leaves by up to 60-fold and in roots by 20-fold; as Figures 8 - 9 can be seen, inserting the 4×NRE sequence into the upstream regulatory sequence of GRF4 increased its expression level in leaves by up to 6000-fold and in roots by 400-fold under the same two nitrogen conditions; the fold increase in GRF4 expression level is very high, probably because its original expression level in leaves and roots is very low. These results demonstrate that targeted insertion of the 4×NRE sequence into the upstream regulatory sequences of GRF4 and GS1;2 effectively increased their expression levels.

[0127] Example 4 Agronomic trait evaluation

[0128] 4.1 Experimental design

[0129] Homozygous individuals of three families GRF4-NRE#1, GRF4-NRE#7, and GRF4-NRE#8 with 4×NRE inserted into the GRF4 promoter region and homozygous individuals of three families GS1;2-NRE#1, GS1;2-NRE#7, and GS1;2-NRE#24 with 4×NRE knocked into the GS1;2 promoter region were subjected to agronomic trait evaluation; control: unedited Ningjing 7 (N7).

[0130] The planting time and location are as follows: Nanjing from May to October 2024; Sanya, Hainan from December 2024 to April 2025;

[0131] The measured indexes include: plant height, panicle length, number of effective tillers, number of grains per panicle, 1000-grain weight;

[0132] Plant height: Measure the highest panicle of a single plant, that is, from the base of the stem to the top of the panicle excluding the awn;

[0133] Panicle length: Measure the length of the rice panicle with a ruler, the longest distance from the tip of the panicle to the base of the panicle;

[0134] Number of effective tillers: Count the tillers that produce panicles and set seeds at maturity for each plant;

[0135] Number of grains per panicle: Select seeds in the air-dried state, count all the filled grains and empty grains on a panicle, and the sum is the number of grains per panicle;

[0136] 1000-grain weight: Randomly pick 50 - 60 normal rice seeds into a seed-counter, remove the shrivelled grains and branches, and output the data after confirming the number recognized by the machine.

[0137] 4.2 Data statistics

[0138] The phenotype of GRF4-NRE#8 is as Figure 10 shown, and the number of effective tillers, number of grains per panicle, 1000-grain weight of the GRF4-NRE family are as Figures 11 - 13 shown; the phenotype of GS1;2-NRE#24 is as Figure 14 shown, and the number of effective tillers, number of grains per panicle, 1000-grain weight of the GS1;2-NRE family are as Figures 15 - 17 shown.

[0139] The statistical results of the number of effective tillers, number of grains per panicle, 1000-grain weight of the GRF4-NRE family and the GS1;2-NRE family are shown in Table 3 below.

[0140] Table 3

[0141] Character GRF4-NRE Family GS1;2-NRE Family Control Effective Tillering Number 8.8±1.2* 7.4±0.8 7.2±0.7 Number of Grains per Panicle 305±12** 283±9* 232±8 1000-Grain Weight (g) 28.5±0.6* 27.8±0.5 26.2±0.4

[0142] Note: *P<0.05, **P<0.01, t-test.

[0143] From Figure 10 and Figure 14 it can be seen that compared with the control NingJing7, the phenotypes of GRF4-NRE#8 and GS1;2-NRE#24 have been improved to a certain extent; from Figures 11 - 13It can be seen that the average number of effective tillers in the GRF4-NRE family is 8.8, while that of the control NingJing7 is 7.2, an increase of 20.8%; the average number of grains per panicle in the GRF4-NRE family is 305, while that of the control NingJing7 is 232, an increase of 31.5%; the 1000-grain weight of the GRF4-NRE family is on average 28.5 g, while that of the control NingJing7 is 25.4 g, an increase of 12.2%; the average number of effective tillers in the GS1;2-NRE family is 7.4 g, an increase of 1.44% compared to the control NingJing7; the average number of grains per panicle in the GS1;2-NRE family is 283, an increase of 22.0% compared to the control NingJing7; the 1000-grain weight of the GS1;2-NRE family is on average 27.8 g, an increase of 6.1% compared to the control NingJing7. It shows that inserting 4×NRE into the promoter regions of GRF4 and GS1;2 significantly improves the agronomic traits such as the phenotype, the number of effective tillers, the number of grains per panicle, and the 1000-grain weight of the regenerated rice plants.

[0144] In summary, in this application, by inserting a tandem sequence of 4 cis-acting elements (4×NRE) into the promoter regions of 2 important nitrogen metabolism-related genes (GRF4, GS1;2) to activate gene expression under nitrate induction, thereby enhancing the nitrogen utilization of rice, that is, enhancing the expression of target genes under the condition of applying nitrogen fertilizer in the field, regenerated rice plants with high nitrogen use efficiency are obtained; through dual-luciferase reporter experiments, it is verified that the inserted 4×NRE can significantly enhance the expression of downstream genes, especially in the presence of nitrate; and when the insertion position of 4×NRE is within the range of 100 bp - 400 bp near the transcription start site, the gene activation effect is the best, and the expression level increases by up to 60 times (GS1;2) to 6000 times (GRF4); the transcriptional levels of GRF4 and GS1;2 in the obtained regenerated rice plants are significantly increased under both low-nitrogen and high-nitrogen conditions, indicating an enhanced nitrogen response ability; field experiments show that the number of effective tillers, the number of grains per panicle, and the 1000-grain weight of the regenerated rice plants are significantly increased. Among them, the number of effective tillers in the GRF4-NRE family is increased by 20.8%, the number of grains per panicle is increased by 31.5%, and the 1000-grain weight is increased by 12.2%; the number of grains per panicle in the GS1;2-NRE family is increased by 22.0%, and the 1000-grain weight is increased by 6.1%; the method of this application provides a new strategy for high-nitrogen-efficient rice breeding and can be directly applied to cultivated varieties (such as Ningjing 7), shortening the breeding cycle.

[0145] The above are only optional embodiments of this application, and do not limit the patent scope of this application accordingly. Any equivalent structural transformation made under the inventive concept of this application by using the content of the specification and drawings of this application, or directly / indirectly applied in other related technical fields, is included in the patent protection scope of this application.

Claims

1. A gene editing method for regulating nitrogen use efficiency in rice, characterized in that, Comprising the following steps: Selecting a key gene of rice nitrogen metabolism as a target gene; Determining a target site in the promoter region of the target gene; Based on the sequences on both sides of the target site, constructing a repair donor template containing a nitrate response element; wherein, the nitrate response element is derived from the promoter region of rice nitrite reductase and can respond to nitrate signals to enhance the expression of downstream genes; After co-delivering the repair donor template and the CRISPR / Cas9 vector to rice callus by gene editing, culturing; After the culturing is completed, screening and obtaining regenerated rice plants with the nitrate response element inserted into their genomes.

2. The gene editing method for regulating rice nitrogen use efficiency according to claim 1, wherein In the step of selecting a key gene of rice nitrogen metabolism as a target gene, the target gene is at least one of the GRF4 target gene and the GS1;2 target gene.

3. The gene editing method for regulating rice nitrogen use efficiency according to claim 2, wherein, The gene sequence of the promoter region of the GRF4 target gene is shown as Seq ID NO.3; the gene sequence of the promoter region of the GS1;2 target gene is shown as Seq ID NO.

4.

4. The gene editing method for regulating rice nitrogen use efficiency according to claim 1, characterized in that The step of determining a target site in the promoter region of the target gene includes: Using a dual-luciferase reporter gene assay, segmentally cloning the promoter fragment of the target gene, constructing multiple reporter gene vectors with the nitrate response element inserted at different positions, verifying the expression enhancement effect through protoplast transformation and nitrate induction experiments, and determining the position that can enhance the expression of downstream genes as the target site; Wherein, the target site is within the range of 0.1 kb - 1.0 kb near the transcription start site of the target gene.

5. The gene editing method for regulating rice nitrogen use efficiency according to claim 1, wherein, The nitrate response element is 4 tandem copies of the NRE cis-acting element, and the gene sequence of the nitrate response element is shown as Seq ID NO.

1.

6. The gene editing method for regulating rice nitrogen use efficiency according to claim 1, characterized in that In the step of constructing a repair donor template containing a nitrate response element based on the sequences on both sides of the target site, the homologous arms of the repair donor template match the sequences on both sides of the target site, the repair donor template uses a chemically modified linear DNA fragment, and the repair donor template is prepared by amplifying with a pair of primers with 5' phosphate and terminal 3-base thiophosphate modification.

7. The gene editing method for regulating rice nitrogen use efficiency according to claim 1, wherein In the step of co-delivering the repair donor template and the CRISPR / Cas9 vector to rice callus by gene editing, the CRISPR / Cas9 vector is obtained by connecting the Ubiquitin promoter - Cas9 protein - Nos terminator, the U3 promoter - sgRNA, and the hygromycin resistance screening marker gene in series by the GoldenGate method to the pCam1300 vector backbone.

8. The gene editing method for regulating rice nitrogen use efficiency according to claim 7, characterized in that, The gene sequence of the CRISPR / Cas9 vector is shown as Seq ID NO.

2.

9. The gene editing method for regulating nitrogen use efficiency of rice according to claim 1, wherein In the step of co-delivering the repair donor template and the CRISPR / Cas9 vector to rice callus by gene editing, the repair donor template and the CRISPR / Cas9 vector are co-delivered to rice callus by the gene gun transformation method; The parameters of the gene gun transformation method include: the gold particle size is 0.5 μm - 0.7 μm; the helium pressure is 1100 psi - 1350 psi; the target distance is 5 cm - 7 cm; the CRISPR / Cas9 vector and the repair donor template are mixed at a molar ratio of 1:(5 - 10).

10. A regenerated rice plant obtained by the gene editing method for regulating rice nitrogen use efficiency according to any one of claims 1-9, characterized in that, In the genome of the regenerated rice plants, the promoter region of the GRF4 gene and / or the promoter region of the GS1;2 gene contains an inserted nitrate response element.

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